Conventional methods for transforming monocotyledons include electroporation method, polyethylene glycol method (PEG method), particle gun method and so on.
The electroporation method is a method in which protoplasts and the desired DNA are mixed, and holes are formed in the cell membranes by electric pulse so as to introduce the DNA into the cells, thereby transforming the cells. Various genes have been introduced into monocotyledons, especially into rice plants by this method (Toriyama K. et al., 1988; Biotech. 6:1072-1074, Shimamoto K. et al., 1989; Nature 338:274-276, Rhodes C. A. et al., 1988; Science 240:204-207). However, this method has problems in that 1) it can be applied only to the plant species for which the system for regenerating plants from protoplasts has been established, 2) since it takes several months to regenerate plants from the protoplasts, a long period of time is required to obtain transformants, and 3) since the culture period is long, the frequency of emergence of mutants during the culture is high accordingly, so that the frequency of obtaining normal transformants is decreased.
The PEG method is a method in which the desired gene and protoplasts are mixed and the mixture is treated with PEG, thereby introducing the gene into the protoplasts. This method is different from the electroporation method in that PEG is used instead of the electric pulse. The efficiency of introducing the gene by this method is thought to be somewhat lower than that by the electroporation method. Although there are some reports mentioning that transformants were obtained by this method, this method is not widely used. As using protoplasts, this method has the same problems as in the electroporation method (Zhang W. et al., 1988; Theor. Appl. Genet. 76:835-840, Datta S. K. et al., 1990; Biotech. 8:736-740).
Recently, there has been a report of a method for introducing a gene into immature embryos weakly treated with a cell wall degrading enzyme and calli of maize by electric pulse (D'Halluin K. et al., 1992; Plant Cell 4:1495-1505). The existence of the introduced gene has been confirmed also in the regenerated plants. However, only one report that has disclosed the success in transformation has been made.
The particle gun method is a method in which the desired gene is attached to fine metal particles, and the metal particles are shot into cells or tissues at a high speed, thereby carrying out the transformation. Thus, according to this principle, transformation may be performed on any tissues. Therefore, it is said that this method is effective in transforming the plant species for which the systems for regenerating plants from protoplasts have not been established.
There have been made some reports of obtaining transformants of maize with normal fertility by transforming type II calli of maize (Armstrong C. L. and Green C. E., 1985; Planta 164:207-214) by the particle gun method (Gordon-Kamm W. J. et al., 1990; Plant Cell 2:603-618, Fromm M. E. et al., 1990; Biotech. 8:833-839, Walters D. A. et al., 1992; Plant Mol. Biol. 18:189-200, Vain P. et al., 1993; Plant Cell Rep. 12:84-88). However, almost all these reports used easily-culturable varieties as the starting materials and the techniques disclosed therein could not be applied to any unlimited varieties.
Vasil et al. obtained Basta-resistant calli and regenerated plants by introducing bar gene (Thompson C. J. et al., 1987; EMBO J. 6:2519-2523) capable of acetylating phosphinothricin, which is the main component in herbicides such as Basta, bialaphos, etc., and GUS gene into embryogenic calli of wheat by the use of a particle gun. They identified the activity of the enzyme which is a product from the introduced genes in these calli and regenerated plants and also identified the bar gene in them by Southern blot analysis (Vasil V. et al., 1992; Biotech. 10:667-674).
Li et al. obtained hygromycin-resistant, regenerated plants by introducing a hygromycin-resistant gene into immature embryos and embryogenic calli of rice by the use of a particle gun followed by selecting the transformants. They identified the hygromycin-resistant gene in the plants by Southern blot analysis. They revealed that the segregation ratio of the hygromycin-resistant and hygromycin-sensitive plants in the R1 progeny of the plants was 3:1 (Li L. et al., 1993; Plant Cell Rep. 12:250-255).
Christou et al. obtained plants which are resistant to hygromycin or bialaphos and which have a GUS activity by introducing bar gene, a hygromycin-resistant gene and GUS gene into immature embryos of rice by the use of a particle gun, and they identified the introduced genes in the plants by Southern blot analysis (Christou P. et al., 1991; Biotech 9:957-962).
Koziel et al. obtained phosphinothricin-resistant plants by introducing bar gene and a Bt toxin-producing gene into immature embryos of maize by the use of a particle gun. They identified the production of a protein of Bt toxin in these plants and also the introduced genes therein by Southern blot analysis (Koziel M. G. et al., 1993; Biotech. 11:194-200).
Other methods include 1) culturing seeds or embryos with DNA (Topfer R. et al., 1989; Plant Cell 1:133-139; Ledoux L. et al., 1974; Nature 249:17-21), 2) treatment of pollen tubes (Luo and Wu, 1988; Plant Mol. Biol. Rep. 6:165-174), and 3) liposome method (Caboche M., 1990; Physiol. Plant. 79:173-176, Neuhaus G. et al., 1987; Theor. Appl. Genet. 75:30-36). However, these methods have problems in the efficiency of transformation, reproducibility or applicability, so that these methods are not popular.
On the other hand, a method for introducing a gene using the Ti plasmid of bacteria belonging to genus Agrobacterium as a vector is widely used for transforming dicotyledons such as tobacco, petunia, rape and the like. However, it is said that the hosts for the bacteria belonging to the genus Agrobacterium are restricted to only dicotyledons and that monocotyledons are not infected by Agrobacterium (De Cleene M., 1976; Bot. Rev. 42:389-466).
As for transformation of monocotyledons by Agrobacterium, although transformation of asparagus (Bytebier B. et al., 1987; Proc. Natl. Acad. Sci. USA, 84:5345-5349) and of Dioscore bulbifera (Schafer et al., 1987; Nature 327:529-532) have been reported, it is said that this method cannot be applied to other monocotyledons, especially to the plants belonging to the family Gramineae (Potrykus I., 1990; Biotechnology 8:535-543).
Grimsley et al. reported that T-DNA of Agrobacterium in which DNA of maize streak virus had been inserted was inoculated to the apical meristems of maize plants and infection of the plants by maize streak viruses was confirmed. Since the infected symptoms are not observed when merely the DNA of maize streak virus is inoculated thereto, they interpreted the above-mentioned result as a piece of evidence showing that Agrobacterium can introduce the DNA into maize (Grimsley et al., 1987; Nature 325:177-179). However, since it is possible that viruses replicate even if they are not incorporated into the nucleus genome, the result does not show that the T-DNA was incorporated into the nucleus. They subsequently reported that the infection efficiency is the highest when the Agrobacterium is inoculated to the apical meristems in the shoot apices of the maize (Grimsley et al., 1988; Biotech. 6:185-189), and that virC gene in the plasmid of Agrobacterium is indispensable to the infection (Grimsley et al., 1989; Mol. Gen. Genet. 217:309-316).
Gould et al. inoculated the apical meristems of maize with super-virulent Agrobacterium EHA1 having a kanamycin-resistant gene and GUS gene after having injured them with a needle, and selected the thus-treated apical meristems based on their resistance to kanamycin. As a result, plants having resistance to kanamycin were obtained. They confirmed by Southern blot analysis that some of the seeds of the subsequent generations of the thus-selected plants had the introduced genes (Gould J. et al., 1991; Plant Physiol. 95:426-434). This means that the plants grown from the Agrobacterium-treated apical meristems and selected on the basis of their resistance to kanamycin have both the transformed cells and non-transformed cells (chimera phenomenon).
Mooney et al. tried to introduce a kanamycin-resistant gene into embryos of wheat using Agrobacterium. The embryos were treated with an enzyme to injure their cell walls, and then cells of Agrobacterium were inoculated thereto. Among the treated calli, a very small amount of calli which are assumed to have resistance to kanamycin grew, but plants could not be regenerated from these calli. The existence of the kanamycin-resistant gene in them was checked by Southern blot analysis. As a result, in all of the resistant calli, the change in the structure of the introduced gene was observed (Mooney P. A. et al., 1991; Plant Cell, Tissue, Organ Culture, 25:209-218).
Raineri et al. inoculated 8 varieties of rice with super-virulent Agrobacterium A281 (pTiBo542) after having injured the scutella of the rice plants. As a result, the growth of tumor-like tissues was observed in two varieties, Nipponbare and Fujisaka 5. Further, cells of Agrobacterium containing a plasmid having a T-DNA from which a hormone-synthesizing gene had been removed and instead, a kanamycin-resistant gene and GUS gene had been inserted thereinto were inoculated to the embryos of rice. As a result, the growth of kanamycin-resistant calli was observed. Although the expression of the GUS gene was observed in these resistant calli, transformed plants could not be obtained from the calli. They interpreted from these results that the T-DNA of Agrobacterium was introduced into the rice cells (Raineri et al., 1990; Biotech. 8:33-38).
Thus, the experimental results which suggest that the introduction of genes into the plants belonging to the family Gramineae such as rice, maize and wheat can be attained by using Agrobacterium have been reported. However, all of these have a problem in the reproducibility and gave no convincing results since they did not fully identify the introduced genes (Potrykus I. 1990; Biotech. 8:535-543).
Chan et al. injured immature embryos of rice that had been cultured for 2 days in the presence of 2,4-D and then inoculated thereto cells of Agrobacterium having nptII gene and GUS gene in a medium containing potato suspension cultured cells. They cultured the thus-inoculated immature embryos on a G418-added medium to obtain regenerated plants from the induced calli. They investigated the existence of the GUS gene in the regenerated plants and these progeny by Southern blot analysis and found the existence of the introduced gene both in the R0 and R1 generations (Chan M. T. et al., 1993; Plant Mol. Biol., 22:491-506). These results support the transformation of rice with Agrobacterium but the frequency of transformation was as low as 1.6%. In addition, only one regenerated plant that had normally grown was obtained from the 250 immature embryos tested. The separation of immature embryos from rice plants needs much labor. Therefore, such a low transformation efficiency is not in a practical level.